Changes in DNA accessibility at specific loci in chromatin are important determinants for the regulation of gene activity. The proposed research project will evaluate novel molecular probes capable of detecting changes in DNA accessibility at the single molecule level. The probes will be assembled at specific loci in chromatin, and will generate signals based on bioluminescence. We will construct a novel split luciferase system, which will generate a photon flux high enough to enable single molecule detection. Using mammalian cell culture systems and appropriate DNA transfection vectors, we will evaluate the performance of the molecular probe designs for the purpose of monitoring, though in vivo imaging, changes in chromatin accessibility of specific loci in the genome, as cells undergo normal developmental processes. Evaluation criteria will include signal/noise, signal persistence, spatial resolution, time resolution, and robustness for use in routine biological experiments.